The mixed-function oxidase system includes cytochromes P-450 that metabolize a variety of drugs and carcinogens. The multiple forms of this enzyme display broad, overlapping substrate specificity. The type and amount of each form varies among species and individuals. The focus of this project is the identification, characterization, and elucidation of structure-function relationships of these isozymes. Monoclonal antibodies (MAbs) to specific sytochromes P-450 are an essential tool in these studies. Several cytochromes P-450 have been substantially purified in a one-step immunoadsorption procedure using Sepharose-bound MAbs to the major forms of rat liver cytochrome P-450 induced by 3-methylcholanthrene and phenobarbital (MC-P-450, and PB-P-450, respectively). When mixed with solubilized rat liver microsomes, the immunoadsorbent based on MAb 1-7-1 to MC-P-450 binds two polypeptides of MW 56,000 and MW 57,000, while the immunoadsorbent made with MAb 1-31-2 to MC-P-450 binds only the MW 57,000 species. An immunoadsorbent with the MAb 2-66-3 to PB-P-450 adsorbs a species of MW 54,000. These polypeptides are readily desorbed by 0.1 M glycine (pH 3.0). Additional cytochromes P-450 have been purified with MAbs 1-7-1 and 1-31-2 from livers of C57B1/6 and DBA/2 mice, guinea pigs and hamsters and from rat lung. Such immunoadsorption experiments based on different MAbs reveal epitope relatedness between cytochromes P-450 in different tissues, strains, and species. Cytochromes P-450 isolated by this procedure, although enzymatically inactive, can be further analyzed structurally by peptide mapping and amino acid analysis. Similarities as well as differences in the primary structure of several P-450s were found. In addition we are developing methods for elution of catalytically active cytochrome P-450 from immunoadsorbents.